The national epidemic of amphetamine abuse has prompted NIDA to declare the development of new approaches (e.g., pharmacotherapies) for treating methamphetamine addiction as one of its primary goals. Recent evidence points to the neurotensin receptor system (the GPCRs NTR1 and NTR2) as a potential means of addressing this problem. Despite being discovered over 30 years ago, nonpeptide small-molecule compounds for NT receptors were virtually unknown. Using an SAR scanning library strategy backed up by findings from point mutation studies, we were able to identify small-molecule agonist and selectivity trends starting from nonselective small-molecule antagonists. Our primary assays for the collection of preliminary data included a FLIPR assay for measuring intrinsic activity for the NTR1 receptor and a radioligand binding assay backed by Western blot analysis for NTR2 receptor. We have since developed a FLIPR assay for the NTR2 receptor and will use this in place of radioligand binding and Western blots. Secondary assays are provided by Drs. Caron and Barak who employ high content cell imaging of 2-arrestin/Green Fluorescent Protein (GFP) chimeric fusions which provide a reliable and sensitive means to recognize the agonist-mediated activation states of GPCRs. To identify novel scaffolds to develop into NT receptor small-molecule compounds, our collaborator Dr. Danni L. Harris has constructed a 3D-pharmacophore model using structural and assay data provided by our studies that we are using to screen vast commercial libraries of existing compounds in silico. The numerous hits obtained in this effort are being parsed and filtered to create a list to be screened. We will also identify novel scaffolds for optimization through our collaboration with the Molecular Libraries Probe Production Centers Network which is preparing to screen their 300,000 compound collection using the 2- arrestin assay. We are proposing to use a focused library synthesis/screening iterative process of discovery to optimize the trends discovered in our initial work. We will also apply this same method to the novel scaffolds identified in our in silico screening and Molecular Libraries Probe Production Centers screening efforts to identify and optimize small-molecule compounds that will be useful tools for studying the role of the NT receptor system in addiction to methamphetamine.